Abstract
GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators has been challenging due to paucity of convenient assays. In this study, we describe a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Since Mg2+ plays a critical role in influencing the affinity of GTPases with guanosine triphosphate/guanosine diphosphate (GTP/GDP) and the process of nucleotide exchange, manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP that remains after the GTPase reaction to ATP and detection of the generated ATP using the luciferin/luciferase combination. The GTPase/GAP/GEF-Glo assay system enables monitoring of GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins when expressed in cells as fusion proteins by performing the assay in a pulldown format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds and its robustness was demonstrated by a high Z′-factor of 0.93 and CV of 2.2%. The assay system has a high dynamic range, formatted in a convenient add–mix–read, and applicable to high-throughput screening.
Highlights
Small GTPases are typically 20–25 kDa in size that shuttle between an active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations
The ON–OFF cycle is regulated by two other classes of proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).[8,9]
1,530 of NF1 protein), RhoA, Rab5A, Ran, RCC1, and RapGAP (Rna1p, GAP for yeast ortholog of mammalian Ran GTPase) were obtained from Jena Biosciences GmbH ( Jena, Germany), the Ras protein was obtained from Millipore (Billerica, MA), the Rheb protein and guanosine-S0 [(b,g)-methylano] triphosphate (GMP-PCP) were obtained from Sigma-Aldrich
Summary
Small GTPases are typically 20–25 kDa in size that shuttle between an active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations. The Ras family GTPases mediate signals emanating from cell surface receptors and culminating in transcription, cellular differentiation, and proliferation. The Rho family GTPases regulate cell shape and cytoskeletal processes like cell division and cell migration. GTPases have very high affinity for both GTP and GDP with a Kd in the picomolar to nanomolar range.[4,5] As a result, cellular GTPases are always present in a nucleotide-bound form and rarely in a nucleotidefree state. The active GTP-bound form of GTPases interacts with downstream effector proteins culminating in modulation of cellular signaling. The ON–OFF cycle is regulated by two other classes of proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).[8,9] In a resting cell, the GTPases are in their inactive GDP-bound form. BIOLUMINESCENT GTPASE ASSAY formatted in a convenient add–mix–read format with a high dynamic range, and is ideal for high-throughput screening
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